In addition to low molecular weight nutrients, such as vitamins or metabolites, all cells require a medium supplemented with macromolecular growth factors for proliferation in vitro. Tumorigenic cells are often thought to require fewer such factors than do their normal counterparts. The object of this project is to characterize these nutritional alterations and to determine if such in vitro alterations reflect basic regulatory changes involved in oncogenesis. We have shown, using cells cultured in a chemically defined serum-free medium, that SV40-transformed variants of the rat embryo cell line REF52 have partially obviated the requirement for mitogenic or progression factors, such as epidermal growth factor, for clonal proliferation. Further, tumorigenic sublines of these transformants, as well as nude mouse tumor-derived lines, no longer require any mitogenic factors. We will extend these studies to REF52 cells transformed by human adenovirus type 5, Kirsten sarcoma virus, and polyoma virus to determine the generality of these observations. The complete obviation of mitogenic requirements precedes rather than accompanies the acquisition of oncogenic growth potential for REF52. Rather, a good correlate of oncogenic transformation is the development of the transformed phenotype in serum and mitogen-free medium. Molecular mechanisms for this mitogen-free phenotype, which includes soft-agar growth and loss of actin cables, will be sought. EGF receptor levels, sensitivity to EGF and the role of cellular-transforming factors will all be evaluated in this respect. REF52 cells enter a true Go state when growth-arrested. Only the influence of competence factors will relieve this arrest. The relationship between the ability to enter Go and the phenomenon of cellular immortalization will be studied using REF52 cells transformed by E1A alone of Ad5, a segment of DNA which will immortalize but not transform cells.